Method for detecting aminoethanenitrile in nicorandil bulk drug

Abstract

A method for detecting aminoethanenitrile in nicorandil bulk drug, which adopts hydrophilic octadecyl bonded silica gel chromatographic column, 10-50 mmol / L ammonium acetate buffer solution with pH=2.5-4.0 as mobile phase A, acetonitrile as mobile phase B, ELSD detector, 90-95% of mobile phase A and 5-10% of mobile phase B as initial mobile phase for elution, 5-20% of mobile phase A and 80-95% of mobile phase B for elution after the elution of aminoethanenitrile, and the initial mobile phase for balance after no more chromatographic peaks are eluted. In the method, the elution of aminoethanenitrile does not interfere with the elution of impurity A, impurity B and impurity C of nicorandil in the European Pharmacopoeia, and has good sensitivity, accuracy and durability, low use cost, and good repeatability of detection results, which ensures the controllability of the quality of nicorandil bulk drug.

Description

Technical Field

[0001] This invention belongs to the field of active pharmaceutical ingredient (API) testing technology, specifically relating to a method for detecting aminoethylnitrate in nicorandil API. Background Technology

[0002] Nicorandil is a potassium channel opener that dilates coronary vessels, sustainably increases coronary blood flow, and inhibits coronary artery spasm. Nicorandil also exhibits nitrate-like effects, providing a strong vasodilatory effect on large coronary arteries and further increasing coronary blood flow. Therefore, nicorandil can improve myocardial ischemia through a dual mechanism of action.

[0003] Recent studies have found that nicorandil can reduce myocardial damage caused by ischemia / reperfusion, decrease myocardial edema and infarct size, and also inhibit platelet aggregation to prevent thrombosis. As one of the first-line treatments for myocardial ischemia, nicorandil can improve coronary microcirculation in patients. It is also used as an antianginal drug to treat angina.

[0004] According to the publicly available synthetic route of nicorandil, the synthesis of nicorandil from nicotinic acid inevitably involves the use of large and excessive amounts of ethanolamine and nitric acid. Ethanolamine reacts with nitric acid in the nitration reaction to produce the impurity acetic acid, as shown in the following reaction formula:

[0005] In the process of condensing nicotinic acid derivatives, such as nicotinic acid esters, nicotinic acid hydrazide, nicotinic acid imidazole, and 3-pyridaldehyde, with aminoethyl nitrate to obtain nicotinic acid, aminoethyl nitrate is present in the reaction system as one of the raw materials. This impurity does not have a typical genotoxicity warning structure, and according to ICH guidelines, it is controlled as another individual impurity, with a content not exceeding 0.10%. Typically, this impurity needs to be removed during the purification process. To verify the effectiveness of the purification process in removing impurities, a detection method for this impurity needs to be developed to detect the aminoethyl nitrate content in nicotinic acid raw material and intermediates, without interference from known controlled impurities.

[0006] However, nitric acid (aminoethyl nitrate) lacks a chromogenic group and does not absorb ultraviolet light. For the detection of substances without ultraviolet absorption, broad-spectrum detectors such as refractive index (RID) and electrospray ionization (CAD) detectors are typically used. RID offers advantages such as wide applicability and ease of use, but its low sensitivity makes it difficult to meet the needs of detecting trace impurities, and it cannot utilize gradient elution, limiting the flexibility of method development. CAD, on the other hand, is expensive, making it difficult for most laboratories to meet their requirements. Currently, no methods for detecting impurities without ultraviolet absorption in nicotinic acid have been reported in the market. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting aminoethyl nitrate in nicorandil raw material. The method uses an evaporative light scattering detector (ELSD), and the peaks of aminoethyl nitrate do not interfere with those of controlled impurities A, B, and C in the European Pharmacopoeia. It has good sensitivity, accuracy, and durability, low cost, and good repeatability of detection results, thus ensuring the controllability of nicorandil raw material quality.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for detecting acetaminophen in nicorandil raw material, comprising the following steps: The nicorandil raw material test solution was detected by high performance liquid chromatography (HPLC). A hydrophilic octadecyl bonded silica column was used. The mobile phase A was 10-50 mmol / L ammonium acetate buffer with pH 2.5-4.0. The pH was adjusted with acetic acid or formic acid. Gradient elution was performed using acetonitrile as the mobile phase B. The detection was performed by evaporative light scattering (ELSD). The ELSD nebulizer temperature and evaporator temperature were both 40-70℃.

[0009] Furthermore, the gradient elution procedure is as follows: using mobile phase A (90-95% by volume) and mobile phase B (5-10% by volume) as the starting mobile phase, perform isocratic elution for 7-15 minutes. After the chromatographic peaks of ammonia, acetyl nitrate, and nitric acid are completely eluted, perform isocratic elution for 10-20 minutes using mobile phase A (5-20% by volume) and mobile phase B (80-95% by volume). After no more chromatographic peaks are eluted, return to the starting mobile phase for equilibration.

[0010] Furthermore, the nicorandil active pharmaceutical ingredient contains impurity A, impurity B and / or impurity C, wherein impurity A is 2-(isonicotinamide) ethyl nitrate, impurity B is N-(2-hydroxyethyl)-3-pyridinecarboxamide, and impurity C is 2-aminoethyl-pyridine-3-carboxylic acid ester.

[0011] Preferably, the chromatographic column is selected from the Agilent SB AQ series or the YMC PACK AQ series.

[0012] Furthermore, the chromatographic column packing material has a particle size ≤ 5 μm, a length ≥ 15 cm, and an inner diameter ≤ 4.6 mm.

[0013] Furthermore, the temperature of the evaporative light scattering detector (ELSD) atomizer is 60–70°C, and the temperature of the evaporator is 60–70°C.

[0014] Furthermore, the content of nicorandil raw material in the test solution is 100-120 mg / ml.

[0015] Preferably, the pH value of the mobile phase A is adjusted with acetic acid or formic acid.

[0016] Furthermore, during detection, the ELSD carrier gas flow rate was: nitrogen 0.8–2.0 L / min; the column temperature was: 25℃–40℃; and the mobile phase flow rate was: 0.8–1.2 ml / min.

[0017] Preferably, the isocratic elution time of the initial mobile phase is 10 min. After the chromatographic peak of aminoethyl nitrate is eluted, the mobile phase is eluted for 20 min with 20% mobile phase A and 80% mobile phase B, and then returned to the initial mobile phase for equilibration. The equilibration time is >5 min.

[0018] Furthermore, the concentration of ammonium acetate buffer in the mobile phase A is 10–20 mmol / L, the column temperature is 40°C, the ELSD nebulizer temperature is 70°C, the ELSD evaporator temperature is 70°C, the flow rate is 0.8 ml / min, and the ELSD carrier gas flow rate is 2.0 L / min.

[0019] In this invention, an evaporative light scattering detector (ELSD) is used as the detector. Because it is a broad-spectrum detector, it responds to both non-volatile and non-volatile substances. If non-volatile salts such as phosphates are used as buffers, both the buffer salt and the analyte will respond after the mobile phase evaporates, making them difficult to distinguish and potentially preventing the observation of the target chromatographic peak. In this invention, ammonium acetate is used as the buffer salt, and an appropriate concentration range is set to ensure that the peaks of ammonia, ethyl acetate, and nitrate are symmetrical and without tailing. If the concentration of ammonium acetate is too low, the peak shape will be poor; if the concentration is too high, the baseline noise will be high.

[0020] During the preparation of nicorandil raw material, impurities A, B, and C may all be present. Therefore, the development of a method for detecting acetylamine in nicorandil raw material needs to consider the peak patterns of impurities A, B, and C from the European Pharmacopoeia, as well as nicorandil, and avoid peak interference. This invention, through screening of the mobile phase and chromatographic column, uses 90-95% mobile phase A and 5-10% mobile phase B in the initial mobile phase to elute acetylamine within 10 minutes, allowing acetylamine to interact with the impurities. Impurities A, B, and C, as well as nicorandil, can all be separated with good resolution. The chromatographic peaks are symmetrical and sharp, and the precision is good. The recovery rate of acetic acid is between 90.0% and 110.0%, with high accuracy. After the acetic acid peak is eluted, the system is eluted with 5-20% mobile phase A and 80-95% mobile phase B for 10-20 minutes to remove residual peaks. Nicorandil elutes at 17-22 minutes. After no more peaks are eluted, the system is equilibrated.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The analytical method for aminoethylnitrate in nicorandil raw material in this invention is simple to operate, does not interfere with the peaks of impurities A, B, and C, and calculates the specific content of each impurity by adding a correction factor to the principal component self-comparison method. Moreover, the detection results have good repeatability, ensuring the controllability of the quality of nicorandil raw material.

[0022] The detection method of this invention has high accuracy and good separation. Through spiking recovery tests, the recovery rates of acetaminophen at concentrations of 77.96 μg / ml to 116.93 μg / ml are all between 90.0% and 110.0%. The relative standard deviation of the peak area of ​​acetaminophen is less than 5.0%, demonstrating good precision, accuracy, repeatability, and sensitivity. Furthermore, the impurities do not interfere with the detection of acetaminophen, resulting in highly reliable results. This provides a reliable testing basis for the quality testing of nicorandil raw materials. Attached Figure Description

[0023] Figure 1 This is the chromatogram of the test solution in Example 1 of the present invention.

[0024] Figure 2 This is the localization map of impurity A reference standard in Example 1 of the present invention.

[0025] Figure 3 This is the localization map of impurity B reference standard in Example 1 of the present invention.

[0026] Figure 4 This is the localization map of impurity C reference standard in Example 1 of the present invention.

[0027] Figure 5 This is the localization map of the nitric acid reference standard in Example 1 of the present invention.

[0028] Figure 6 This is the system adaptability detection spectrum in Embodiment 1 of the present invention.

[0029] Figure 7 This is the regression linear equation for Embodiment 1 of the present invention.

[0030] Figure 8 This is the detection chromatogram of the test solution in Example 2 of the present invention.

[0031] Figure 9 This is the detection spectrum of the ammonia-ethyl nitric acid solution in Example 3 of the present invention.

[0032] Figure 10 This is the detection spectrum of the ammonia-ethyl nitric acid solution in Example 4 of the present invention.

[0033] Figure 11 This is the detection spectrum of the ammonia-ethyl nitric acid solution in Example 5 of the present invention.

[0034] Figure 12This is the detection spectrum of the ammonia-ethyl nitric acid solution in Example 6 of the present invention.

[0035] Figure 13 The detection chromatogram is shown for the test sample in Comparative Example 1.

[0036] Figure 14 The detection chromatogram is for the test sample in Comparative Example 2. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the scope of the embodiments described herein.

[0038] For experimental methods not specifically described in the following examples, follow conventional methods and conditions, or select according to the product instructions; in the chromatograms of the examples and comparative examples, the unit of peak time is minutes (min).

[0039] Sources of reagents and impurity reference standards used in the embodiments of this invention: Nicorandil raw material, 100.4% purity, Shanghai Xudong Haipu Pharmaceutical Co., Ltd., batch number 210401YL.

[0040] Aminoacetic acid nitrate, purity 90.11%, purchased from Cisco (Shenzhen) Pharmaceutical R&D Co., Ltd., batch number 19-10-0883; Nicorandil EP impurity A, purity 96.33%, purchased from Cisco (Shenzhen) Pharmaceutical R&D Co., Ltd., batch number 19-04-1239; Nicorandil EP impurity B, purity 95.89%, purchased from Cisco (Shenzhen) Pharmaceutical R&D Co., Ltd., batch number 20-01-0802; Nicorandil EP impurity C, purity 95.68%, purchased from Cisco (Shenzhen) Pharmaceutical R&D Co., Ltd., batch number 20-01-0808; unless otherwise specified, purity refers to mass percentage.

[0041] Specific information on ammonia-ethyl nitric acid, impurity A, impurity B, and impurity C is shown in Table 1. Table 1 ; Example 1: System suitability of the method for detecting acetaminophen in nicorandil raw material. The experimental conditions are as follows: High performance liquid chromatograph: Agilent 1260, ELSD detector; Chromatographic column: Agilent SBAQ column, 250mm × 4.6mm, packing particle size 5μm; Mobile phase A: 10 mmol / L ammonium acetate solution, with the pH adjusted to 2.5 using acetic acid; Mobile phase B: Acetonitrile; Column temperature: 40℃; Flow rate: 0.8 ml / min; Injection volume: 10 μl; ELSD atomizer temperature: 70℃; ELSD evaporator temperature: 70℃; ELSD carrier gas flow rate: nitrogen, 2.0 L / min.

[0042] 1. System adaptability testing Diluent: Acetonitrile.

[0043] Preparation of nicorandil raw material test solution: Take an appropriate amount of nicorandil raw material, accurately weigh it, dissolve and dilute it with diluent to prepare a solution containing about 100mg per ml, shake well, and the solution is ready.

[0044] Impurity reference solution: EP Impurity A Reference Solution: Take an appropriate amount of EP Impurity A reference standard, dissolve and dilute it with diluent to prepare a 0.1 mg / ml solution.

[0045] EP Impurity B Reference Solution: Take an appropriate amount of EP Impurity B reference standard, dissolve and dilute it with diluent to prepare a 0.1 mg / ml solution.

[0046] EP Impurity C Reference Solution: Take an appropriate amount of EP Impurity C reference standard, dissolve and dilute it with diluent to prepare a 0.1 mg / ml solution.

[0047] Nitric acid positioning solution: Take an appropriate amount of nitric acid and dilute it with a diluent to prepare a solution of about 0.1 mg / ml.

[0048] Ammonia-acetyl nitrate reference standard: Ammonia-acetyl nitrate·nitrate.

[0049] Preparation of system suitability solution: Take appropriate amounts of aminoethyl nitrate reference standard, nicorandil raw material, and nicorandil EP impurity A, B, and C reference standards, accurately weigh them, dissolve and dilute them with diluent to prepare a solution containing approximately 100 mg of nicorandil and 0.1 mg each of aminoethyl nitrate, nicorandil EP impurity A, EP impurity B, and EP impurity C per ml.

[0050] System suitability requirements: The chromatographic peaks of nicorandil (European Pharmacopoeia) impurities A, B, and C, as well as nicorandil, should not interfere with the chromatographic peak of acetaminophen.

[0051] Since the reference standard for aminoethyl nitrate in the embodiments of the present invention is aminoethyl nitrate·nitrate, nitrate and aminoethyl nitrate will respectively elute in liquid chromatography, and the nitrate positioning solution is used to locate the nitrate.

[0052] Take blank solvent (i.e., acetonitrile diluent), test solution, EP impurity A reference solution, EP impurity B reference solution, EP impurity C reference solution, and nitric acid positioning solution, respectively, and inject them into the high-performance liquid chromatograph for detection. Perform gradient elution according to the gradient shown in Table 2, and record the chromatograms. The chromatograms of the test solution, each impurity reference standard, and the nitric acid positioning solution are shown in Table 2. Figures 1-5 .

[0053] Take 10 μl each of blank solvent (i.e., acetonitrile diluent) and system suitability solution, and inject them into the high-performance liquid chromatograph for detection. Perform gradient elution according to the gradient shown in Table 2, and record the chromatograms. The system suitability chromatogram is shown in [Table 2]. Figure 6 .

[0054] Table 2 Gradient elution program ; Depend on Figures 1-5 It can be seen that the elution time of nitric acid is 5.42 minutes, the elution time of impurities A, B, and C is around 7.17 minutes, the elution time of blank solvent is 18-19 minutes, and the elution time of nicotinic acid is 19-20 minutes. Figure 6 As can be seen, the elution times of impurities A, B, and C are all around 7.17 minutes, while the elution times of acetic acid, nitrate, and nicorandil are around 6.34 minutes, nitrate, and nicorandil are around 5.36 minutes. According to the localization results, nicorandil and all other impurities can be well separated from the acetic acid chromatographic peaks, and the remaining chromatographic peaks do not interfere with the detection of acetic acid, indicating good system adaptability.

[0055] 2. Linear Case Aminoacetic acid reference standard stock solution: Accurately weigh about 5 mg of aminoacetic acid reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well to obtain the solution.

[0056] Aminoacetic acid reference solution 1 (std-1): Measure 0.5 ml of the aminoacetic acid reference solution into a 5 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0057] Aminoacetic acid reference solution 2 (std-2): Accurately measure 0.8 ml of the aminoacetic acid reference solution, place it in a 5 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0058] Aminoacetic acid reference solution 3 (std-3): Accurately measure 1.0 ml of the aminoacetic acid reference solution, place it in a 5 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0059] Aminoacetic acid reference solution 4 (std-4): Accurately measure 1.2 ml of the aminoacetic acid reference solution, place it in a 5 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0060] Aminoacetic acid reference solution 5 (std-5): Accurately measure 1.5 ml of the aminoacetic acid reference solution, place it in a 5 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0061] 10 μl each of the above-mentioned aminoethyl nitrate reference solutions 1 (std-1), 2 (std-2), 3 (std-3), 4 (std-4), and 5 (std-5) were injected into the high-performance liquid chromatograph for detection. Gradient elution was performed according to the gradient shown in Table 2, and the chromatograms were recorded.

[0062] Five concentration points were selected for the study within the range of the limit of quantitation (LOQ) of the aminoacetyl nitrate reference standard (48.72 μg / ml) to not less than 150% of the index concentration. The linear series concentrations included: LOQ concentration, 80% impurity limit concentration, 100% impurity limit concentration, 120% impurity limit concentration, and 150% impurity limit concentration. The linear regression equation was calculated using the logarithm of the concentration of the aminoacetyl nitrate reference standard solution and the logarithm of the corresponding peak area. The correlation coefficient r should not be less than 0.950. The results are shown in Table 3, and the linear regression equation is shown below. Figure 7 .

[0063] Table 3 ; The linear equation is y = 1.6574x + 3.9400; within the range of 48.72 μg / ml to 146.17 μg / ml, the linear regression coefficient r is 0.9896, indicating a good linear relationship.

[0064] Example 2 Chromatographic conditions: Chromatographic column: YMCPACKAQ, 250mm×4.6mm, packing particle size 5μm; Mobile phase A: 10 mmol / L ammonium acetate solution, with pH adjusted to 3.0 using acetic acid; Mobile phase B: Acetonitrile.

[0065] Unless otherwise specified, the other experimental conditions and liquid chromatography methods are as shown in Example 1. The solutions are prepared as shown below, or they can be prepared according to methods known to those skilled in the art.

[0066] Test solution: Take appropriate amounts of aminoethyl nitrate reference standard, nicorandil raw material, and nicorandil EP impurity A, B, and C reference standards, accurately weigh them, dissolve and dilute them with diluent to prepare a solution containing approximately 100 mg of nicorandil and 0.1 mg each of aminoethyl nitrate, nicorandil EP impurity A, EP impurity B, and EP impurity C per ml.

[0067] See the detection spectrum Figure 8 ,Depend on Figure 8 As can be seen, the elution times of impurities A, B, and C are all around 7.00 minutes, while the elution times of acetic acid and nitric acid are 5.94 minutes and 5.46 minutes, respectively. The resolution between acetic acid and nitric acid is 3.55. The target chromatographic peak is sharp and has good symmetry. Nicorandil elutes at 20–22 minutes. Nicorandil and all other impurities can be well separated from the acetic acid chromatographic peak, and the remaining chromatographic peaks do not interfere with the detection of acetic acid.

[0068] Examples 3-6 and Comparative Examples 1-2 Unless otherwise specified, the other experimental conditions and liquid chromatography methods are as shown in Example 1. The solutions are prepared as shown below, or they can be prepared according to methods known to those skilled in the art.

[0069] The ammonia-acetyl nitrate solution in Examples 3-6 and Comparative Examples 1-2: Accurately measure 1.0 ml of the ammonia-acetyl nitrate reference stock solution in Example 1, place it in a 5 ml volumetric flask, add diluent to dilute to the mark, and shake well to obtain the solution.

[0070] According to the detection conditions shown in Table 4, 10 μl of the aminoethyl nitrate reference solution was injected into the high-performance liquid chromatograph for detection. Gradient elution was performed using the gradient shown in Table 2, and the chromatograms were recorded. The results are shown in Table 5. The detection chromatograms of Examples 3-6 are shown in Table 5. Figures 9-12 The detection spectra of comparative examples 1 and 2 are shown below. Figures 13-14 .

[0071] Table 4 ; ELSD temperature refers to the temperature of the atomizer and evaporator.

[0072] Table 5 ; From Table 4-5 and Figure 9-14 As can be seen, under the detection conditions of the present invention, the separation of the chromatographic peaks of aminoethyl nitric acid and the adjacent nitric acid is good, and the peaks are sharp and symmetrical. However, without the addition of ammonium acetate solution, the peaks are severely tailed and have poor symmetry. When the concentration of ammonium acetate solution of the present invention is added to the mobile phase, if the pH is too high or the ELSD temperature is too low, the chromatographic peaks cannot be identified in the spectrum.

Claims

1. A method for detecting acetaminophen in nicorandil raw material, comprising the following steps: The test solution was analyzed by high performance liquid chromatography using a hydrophilic octadecyl bonded silica column. Gradient elution was performed using 10–50 mmol / L ammonium acetate buffer (pH 2.5–4.0) as mobile phase A and acetonitrile as mobile phase B. Detection was performed using an evaporative light scattering detector (ELSD). The ELSD nebulizer temperature and evaporator temperature were both 40–70 °C. The gradient elution program is as follows: use 90-95% mobile phase A and 5-10% mobile phase B as the starting mobile phase and elute isocratically for 7-15 minutes. After the chromatographic peaks of aminoacetic acid and nitrate are completely eluted, use 5-20% mobile phase A and 80-95% mobile phase B as the starting mobile phase and elute isocratically for 10-20 minutes, and then return to the starting mobile phase for equilibration.

2. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, The nicorandil active pharmaceutical ingredient contains impurities A, B and / or C, wherein impurity A is 2-(isonicotinamide) ethyl nitrate, impurity B is N-(2-hydroxyethyl)-3-pyridinecarboxamide and impurity C is 2-aminoethyl-pyridine-3-carboxylic acid ester.

3. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, The chromatographic column is selected from the Agilent SBAQ series or YMCPACKAQ series. The particle size of the chromatographic column packing is ≤5μm, the length is ≥15cm, and the inner diameter is ≤4.6mm.

4. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, The temperature of the evaporative light scattering detector (ELSD) atomizer is 60–70°C, and the temperature of the evaporator is 60–70°C.

5. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, The content of nicorandil raw material in the test solution is 100-120 mg / ml.

6. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, In the mobile phase A, the pH value is adjusted with acetic acid or formic acid.

7. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, During detection, the ELSD carrier gas flow rate was: nitrogen 0.8–2.0 L / min; the column temperature was: 25℃–40℃; and the mobile phase flow rate was: 0.8–1.2 ml / min.

8. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, The isocratic elution time of the initial mobile phase is 10 min. After the chromatographic peak of aminoethyl nitrate is eluted, the mobile phase is eluted with 20% mobile phase A and 80% mobile phase B for 20 min, and then returned to the initial mobile phase for equilibration. The equilibration time is >5 min.

9. The method for detecting aminoethylnitrate in nicorandil raw material according to claim 1, characterized in that, The concentration of ammonium acetate buffer in mobile phase A is 10-20 mmol / L, the column temperature is 40℃, the ELSD nebulizer temperature is 70℃, the ELSD evaporator temperature is 70℃, the flow rate is 0.8 ml / min, and the ELSD carrier gas flow rate is 2.0 L / min.

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